Discovery and Characterization of VU0542270, the First Selective Inhibitor of Vascular Kir6.1/SUR2B KATP Channels.

Vascular smooth muscle KATP channels critically regulate blood flow and blood pressure by modulating vascular tone and therefore represent attractive drug targets for treating several cardiovascular disorders. However, the lack of potent inhibitors that can selectively inhibit Kir6.1/SUR2B (vascular KATP) over Kir6.2/SUR1 (pancreatic KATP) has eluded discovery despite decades of intensive research. We therefore screened 47,872 chemically diverse compounds for novel inhibitors of heterologously expressed Kir6.1/SUR2B channels. The most potent inhibitor identified in the screen was an N-aryl-N'-benzyl urea compound termed VU0542270. VU0542270 inhibits Kir6.1/SUR2B with an IC50 of approximately 100 nM but has no apparent activity toward Kir6.2/SUR1 or several other members of the Kir channel family at doses up to 30 µM (>300-fold selectivity). By expressing different combinations of Kir6.1 or Kir6.2 with SUR1, SUR2A, or SUR2B, the VU0542270 binding site was localized to SUR2. Initial structure-activity relationship exploration around VU0542270 revealed basic texture related to structural elements that are required for Kir6.1/SUR2B inhibition. Analysis of the pharmacokinetic properties of VU0542270 showed that it has a short in vivo half-life due to extensive metabolism. In pressure myography experiments on isolated mouse ductus arteriosus vessels, VU0542270 induced ductus arteriosus constriction in a dose-dependent manner similar to that of the nonspecific KATP channel inhibitor glibenclamide. The discovery of VU0542270 provides conceptual proof that SUR2-specific KATP channel inhibitors can be developed using a molecular target-based approach and offers hope for developing cardiovascular therapeutics targeting Kir6.1/SUR2B. SIGNIFICANCE STATEMENT: Small-molecule inhibitors of vascular smooth muscle KATP channels might represent novel therapeutics for patent ductus arteriosus, migraine headache, and sepsis; however, the lack of selective channel inhibitors has slowed progress in these therapeutic areas. Here, this study describes the discovery and characterization of the first vascular-specific KATP channel inhibitor, VU0542270.

Ischemic postconditioning reduces spinal cord ischemia-reperfusion injury through ATP-sensitive potassium channel.

STUDY DESIGN: Animal study. OBJECTIVES: Explore the neuroprotective effect of remote limb ischemic postconditioning (Post C) in spinal cord ischemic reperfusion injury (SCII) and related mechanisms. SETTING: Anesthesiology Laboratory of Southwest Medical University. METHODS: We established a rabbit SCII model and processed it with Post C. To evaluate the neural function, spinal cord tissue was taken 48 h later, normal neurons were evaluated by HE staining, and the expression of ATP-sensitive potassium channel (KATP) marker molecule Kir6.2 was detected by Western blot. Immunofluorescence detection of spinal cord Iba-1 expression, ELISA detection of M1 type microglia marker iNOS and M2 type microglia marker Arg, and Western blot detection of NF-κB and IL-1ß expression. Through these experiments, we will explore the protective effect of Post C in SCII, observe the changes in the protective effect after using KATP blockers, and verify that Post C can play a neuroprotective effect in SCII by activating KATP. RESULTS: We observed that Post C significantly improved exercise ability and the number of spinal motor neurons in the SCII model. Microglia are activated and expression of M1 microglia in the spinal cord was decreased, while M2 was increased. This neuroprotective effect was reversed by the nonspecific KATP inhibitor. CONCLUSION: Post C has a neuroprotective effect on SCII, and maybe a protective effect produced by activating KATP to regulate spinal microglia polarization and improve neuroinflammation.

Mediation of the Cardioprotective Effects of Mannitol Discovered, with Refutation of Common Protein Kinases.

The osmodiuretic agent Mannitol exerts cardioprotection against ischemia and reperfusion (I/R) injury when applied as a pre- and/or postconditioning stimulus. Previously, we demonstrated that these properties are mediated via the activation of mitochondrial ATP-sensitive potassium (mKATP) channels. However, considering Mannitol remains in the extracellular compartment, the question arises as to which receptor and intracellular signaling cascades are involved in myocardial protection by the osmodiuretic substance. Protein kinase B (Akt) and G (PKG), as part of the reperfusion injury salvage kinase (RISK) and/or endothelial nitric oxide (eNOS)/PKG pathway, are two well-investigated intracellular targets conferring myocardial protection upstream of mitochondrial potassium channels. Adenosine receptor subtypes have been shown to trigger different cardioprotective pathways, for example, the reperfusion injury. Further, Mannitol induces an increased activation of the adenosine 1 receptor (A1R) in renal cells conferring its nephroprotective properties. Therefore, we investigated whether (1) Akt and PKG are possible signaling targets involved in Mannitol-induced conditioning upstream of the mKATP channel and/or whether (2) cardioprotection by Mannitol is mediated via activation of the A1R. All experiments were performed on male Wistar rats in vitro employing the Langendorff isolated heart perfusion technique with infarct size determination as the primary endpoint. To unravel possible protein kinase activation, Mannitol was applied in combination with the Akt (MK2206) or PKG (KT5823) inhibitor. In further groups, an A1R blocker (DPCPX) was given with or without Mannitol. Preconditioning with Mannitol (Man) significantly reduced the infarct size compared to the control group. Co-administration of the A1R blocker DPXPC fully abolished myocardial protection of Mannitol. Interestingly and in contrast to the initial hypothesis, neither administration of the Akt nor the PKG blocker had any impact on the cardioprotective properties of Mannitol-induced preconditioning. These results are quite unexpected and show that the protein kinases Akt and PKG-as possible targets of known protective signaling cascades-are not involved in Mannitol-induced preconditioning. However, the cardioprotective effects of Mannitol are mediated via the A1R.

The anti-nociceptive activity of naringenin passes through L-arginine/NO/cGMP/KATP channel pathway and opioid receptors.

As a promising flavonoid, naringenin has shown potential anti-inflammatory and antioxidant properties mainly in inflammatory pain models by oral administration. Therefore, we investigated the antinociceptive activity of this compound by intraperitoneally (i.p.) administration, as well as, associated mechanism of action considering the involvement of L-arginine/nitric oxide (NO)/cyclic GMP (cGMP)/potassium channel (KATP) pathway and opioid receptors. The antinociceptive effect of naringenin was evaluated in male NMRI mice using formalin test at early and late phases. To assess the involvement of L-arginine/NO/cGMP/KATP pathway and opioid receptors, mice were pretreated i.p. with L-arginine (NO precursor), S-nitroso-N-acetylpenicillamine (SNAP, NO donor), N(gamma)-nitro-L-arginine methyl ester (L-NAME, inhibitor of nitric oxide synthase), sildenafil (inhibitor of phosphodiesterase enzyme), glibenclamide (KATP channel blocker) and naloxone (an opioid receptor antagonist), respectively 20 min before administration of the most effective dose of naringenin. Naringenin showed a dose-dependent antinociceptive effect at both early and late phases of the formalin test. The dose of 100 mg/kg of naringenin was identified as the most effective dose and selected for further experiments. Our mechanistic evaluations showed that L-arginine, SNAP and sildenafil could enhance the antinociceptive effects of naringenin, revealing the critical role of NO and cGMP during its antinociceptive effect. On the other hand, glibenclamide and naloxone could mitigate the antinociceptive potential of naringenin at both phases of formalin test, which confirmed the associated role of KATP channels and opioid receptors. In conclusion, naringenin could be a promising antinociceptive agent acting through opioid receptors and L-arginine/NO/cGMP/KATP channel pathway.

Naloxone Protects against Lipopolysaccharide-Induced Neuroinflammation and Microglial Activation via Inhibiting ATP-Sensitive Potassium Channel.

AIM: The aim of this study was to evaluate the anti-inflammatory effects and underlying mechanism of naloxone on lipopolysaccharide- (LPS-) induced neuronal inflammation and microglial activation. METHODS: LPS-treated microglial BV-2 cells and mice were used to investigate the anti-inflammatory effects of naloxone. RESULTS: The results showed that naloxone dose-dependently promoted cell proliferation in LPS-induced BV-2 cells, downregulated the expression of proinflammatory cytokines (TNF-α, IL-1ß, and IL-6) and proinflammatory enzymes iNOS and COX-2 as well as the expression of free radical molecule NO, and reduced the expression of Iba-1-positive microglia in LPS-stimulated BV-2 cells and mouse brain. Moreover, naloxone improved LPS-induced behavior degeneration in mice. Mechanically, naloxone inhibited LPS-induced activation in the ATP-sensitive potassium (KATP) channel. However, the presence of glibenclamide (Glib), an antagonist of KATP channel, ameliorated the suppressive effects of naloxone on inflammation and microglial activation. CONCLUSION: Naloxone prevented LPS-induced neuroinflammation and microglial activation partially through the KATP channel. These findings might highlight the potential of naloxone in neuroinflammation therapy.

KATP channel blockers control glucagon secretion by distinct mechanisms: A direct stimulation of α-cells involving a [Ca2+]c rise and an indirect inhibition mediated by somatostatin.

OBJECTIVE: Glucagon is secreted by pancreatic α-cells in response to hypoglycemia and its hyperglycemic effect helps to restore normal blood glucose. Insulin and somatostatin (SST) secretions from ß- and δ-cells, respectively, are stimulated by glucose by mechanisms involving an inhibition of their ATP-sensitive K+ (KATP) channels, leading to an increase in [Ca2+]c that triggers exocytosis. Drugs that close KATP channels, such as sulfonylureas, are used to stimulate insulin release in type 2 diabetic patients. α-cells also express KATP channels. However, the mechanisms by which sulfonylureas control glucagon secretion are still largely debated and were addressed in the present study. In particular, we studied the effects of KATP channel blockers on α-cell [Ca2+]c and glucagon secretion in the presence of a low (1 mM) or a high (15 mM) glucose concentration and evaluated the role of SST in these effects. METHODS: Using a transgenic mouse model expressing the Ca2+-sensitive fluorescent protein, GCaMP6f, specifically in α-cells, we measured [Ca2+]c in α-cells either dispersed or within whole islets (by confocal microscopy). By measuring [Ca2+]c in α-cells within islets and glucagon secretion using the same perifusion protocols, we tested whether glucagon secretion correlated with changes in [Ca2+]c in response to sulfonylureas. We studied the role of SST in the effects of sulfonylureas using multiple approaches including genetic ablation of SST, or application of SST-14 and SST receptor antagonists. RESULTS: Application of the sulfonylureas, tolbutamide, or gliclazide, to a medium containing 1 mM or 15 mM glucose increased [Ca2+]c in α-cells by a direct effect as in ß-cells. At low glucose, sulfonylureas inhibited glucagon secretion of islets despite the rise in α-cell [Ca2+]c that they triggered. This glucagonostatic effect was indirect and attributed to SST because, in the islets of SST-knockout mice, sulfonylureas induced a stimulation of glucagon secretion which correlated with an increase in α-cell [Ca2+]c. Experiments with exogenous SST-14 and SST receptor antagonists indicated that the glucagonostatic effect of sulfonylureas mainly resulted from an inhibition of the efficacy of cytosolic Ca2+ on exocytosis. Although SST-14 was also able to inhibit glucagon secretion by decreasing α-cell [Ca2+]c, no decrease in [Ca2+]c occurred during sulfonylurea application because it was largely counterbalanced by the direct stimulatory effect of these drugs on α-cell [Ca2+]c. At high glucose, i.e., in conditions where glucagon release was already low, sulfonylureas stimulated glucagon secretion because their direct stimulatory effect on α-cells exceeded the indirect effect by SST. Our results also indicated that, unexpectedly, SST-14 poorly decreased the efficacy of Ca2+ on exocytosis in ß-cells. CONCLUSIONS: Sulfonylureas exert two opposite actions on α-cells: a direct stimulation as in ß-cells and an indirect inhibition by SST. This suggests that any alteration of SST paracrine influence, as described in diabetes, will modify the effect of sulfonylureas on glucagon release. In addition, we suggest that δ-cells inhibit α-cells more efficiently than ß-cells.

Regulation of capillary hemodynamics by KATP channels in resting skeletal muscle.

ATP-sensitive K+ channels (KATP ) have been implicated in the regulation of resting vascular smooth muscle membrane potential and tone. However, whether KATP channels modulate skeletal muscle microvascular hemodynamics at the capillary level (the primary site for blood-myocyte O2 exchange) remains unknown. We tested the hypothesis that KATP channel inhibition would reduce the proportion of capillaries supporting continuous red blood cell (RBC) flow and impair RBC hemodynamics and distribution in perfused capillaries within resting skeletal muscle. RBC flux (fRBC ), velocity (VRBC ), and capillary tube hematocrit (Hctcap ) were assessed via intravital microscopy of the rat spinotrapezius muscle (n = 6) under control (CON) and glibenclamide (GLI; KATP channel antagonist; 10 µM) superfusion conditions. There were no differences in mean arterial pressure (CON:120 ± 5, GLI:124 ± 5 mmHg; p > 0.05) or heart rate (CON:322 ± 32, GLI:337 ± 33 beats/min; p > 0.05) between conditions. The %RBC-flowing capillaries were not altered between conditions (CON:87 ± 2, GLI:85 ± 1%; p > 0.05). In RBC-perfused capillaries, GLI reduced fRBC (CON:20.1 ± 1.8, GLI:14.6 ± 1.3 cells/s; p < 0.05) and VRBC (CON:240 ± 17, GLI:182 ± 17 µm/s; p < 0.05) but not Hctcap (CON:0.26 ± 0.01, GLI:0.26 ± 0.01; p > 0.05). The absence of GLI effects on the %RBC-flowing capillaries and Hctcap indicates preserved muscle O2 diffusing capacity (DO2 m). In contrast, GLI lowered both fRBC and VRBC thus impairing perfusive microvascular O2 transport (Q̇m) and lengthening RBC capillary transit times, respectively. Given the interdependence between diffusive and perfusive O2 conductances (i.e., %O2 extraction∝DO2 m/Q̇m), such GLI alterations are expected to elevate muscle %O2 extraction to sustain a given metabolic rate. These results support that KATP channels regulate capillary hemodynamics and, therefore, microvascular gas exchange in resting skeletal muscle.

Iptakalim alleviates synaptic damages via targeting mitochondrial ATP-sensitive potassium channel in depression.

Synaptic plasticity damages play a crucial role in the onset and development of depression, especially in the hippocampus, which is more susceptible to stress and the most frequently studied brain region in depression. And, mitochondria have a major function in executing the complex processes of neurotransmission and plasticity. We have previously demonstrated that Iptakalim (Ipt), a new ATP-sensitive potassium (K-ATP) channel opener, could improve the depressive-like behavior in mice. But the underlying mechanisms are not well understood. The present study demonstrated that Ipt reversed depressive-like phenotype in vivo (chronic mild stress-induced mice model of depression) and in vitro (corticosterone-induced cellular model). Further study showed that Ipt could upregulate the synaptic-related proteins postsynaptic density 95 (PSD 95) and synaptophysin (SYN), and alleviated the synaptic structure damage. Moreover, Ipt could reverse the abnormal mitochondrial fission and fusion, as well as the reduced mitochondrial ATP production and collapse of mitochondrial membrane potential in depressive models. Knocking down the mitochondrial ATP-sensitive potassium (Mito-KATP) channel subunit MitoK partly blocked the above effects of Ipt. Therefore, our results reveal that Ipt can alleviate the abnormal mitochondrial dynamics and function depending on MitoK, contributing to improve synaptic plasticity and exert antidepressive effects. These findings provide a candidate compound and a novel target for antidepressive therapy.

Aqueous extract of oakmoss produces antihypertensive activity in L-NAME-induced hypertensive rats through sGC-cGMP pathway.

BACKGROUND: Lichens are a symbiotic association of a fungus with a green alga or cyanobacterium. They are widely used in traditional medicine as a treatment against skin disorders, diabetes and hypertension. THE AIM OF THE STUDY: The goal of this paper was to assess the possible antihypertensive and vasorelaxant capacity of the aqueous extract of a lichen species called Oakmoss or Evernia prunastri (L.). MATERIAL AND METHODS: In the present study, the aqueous extract of Oakmoss was prepared, its antihypertensive activity was examined in N(ω)-nitro-L-arginine methyl ester (L-NAME)-induced hypertensive rats, and its vasorelaxant ability was performed in rat isolated thoracic aorta. RESULTS: The results proved that Oakmoss reduced the systolic, diastolic, mean arterial blood pressure, and heart rate in hypertensive rats but not in normotensive rats. Besides, the data showed that Oakmoss exerts its antihypertensive effect through vasorelaxant ability. CONCLUSION: The present study presents the favorable action of Oakmoss as an antihypertensive agent.

Latest Insights into the Pathophysiology of Migraine: the ATP-Sensitive Potassium Channels.

PURPOSE OF REVIEW: Migraine remains a challenging condition to treat, thus highlighting the need for a better understanding of its molecular mechanisms. This review intends to unravel a new emerging target in migraine pathophysiology, the adenosine 5'-triphosphate-sensitive K+ (KATP) channel. RECENT FINDINGS: KATP channel is a common denominator in the cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) mediated intracellular cascades, both of which are involved in migraine. Intravenous infusion of KATP channel opener, levcromakalim, provoked migraine attack associated with dilation of extracerebral arteries in all persons with migraine. Preclinical and clinical studies implicate KATP channels in migraine initiation. KATP channel is a novel therapeutic target for the acute and preventive treatment of migraine. Future studies are warranted to provide a better understanding of the role of KATP channel subgroups in migraine.

ATP-Sensitive Potassium Channels Mediate the Cardioprotective Effect of Panax notoginseng Saponins against Myocardial Ischaemia-Reperfusion Injury and Inflammatory Reaction.

Inflammatory response during myocardial ischemia reperfusion injury (MIRI) is essential for cardiac healing, while excessive inflammation extends the infarction and promotes adverse cardiac remodeling. Understanding the mechanism of these uncontrolled inflammatory processes has a significant impact during the MIRI therapy. Here, we found a critical role of ATP-sensitive potassium channels (KATP) in the inflammatory response of MIRI and its potential mechanism and explored the effects of Panax Notoginseng Saponins (PNS) during this possess. Rats underwent 40 min ischemia by occlusion of the left anterior descending (LAD) coronary artery and 60 min of reperfusion. PNS was treated at the corresponding time point before operation; 5-hydroxydecanoate (5-HD) and glybenclamide (Gly) (or Nicorandil (Nic)) were used as pharmacological blocker (or nonselective opener) of KATP. Cardiac function and pathomorphology were evaluated and a set of molecular signaling experiments was tested. KATP current density was measured by patch-clamp. Results revealed that in MIRI, PNS pretreatment restored cardiac function, reduced infarct size, and ameliorated inflammation through KATP. However, inhibiting KATP by 5-HD and Gly significantly reversed the effects, including NLRP3 inflammasome and inflammatory mediators IL-6, MPO, TNF-α, and MCP-1. Moreover, PNS inhibited the phosphorylation and nuclear translocation of NF-κB in I/R myocardium when the KATP was activated. Importantly, PNS promoted the expression of subunits and activation of KATP. The study uncovered KATP served as a new potential mechanism during PNS modulating MIRI-induced inflammation and promoting injured heart recovery. The manipulation of KATP could be a potential therapeutic approach for MIRI and other inflammatory diseases.

Participation of the opioid receptor - nitric oxide - cGMP - K+ channel pathway in the peripheral antinociceptive effect of nalbuphine and buprenorphine in rats.

The aim of this study was to examine if the peripheral antinociceptive effects of the opioid agonist/antagonist nalbuphine and buprenorphine involve the sequential participation of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) synthesis followed by K+ channel opening in the formalin test. Wistar rats (180-220 g) were injected in the dorsal surface of the right hind paw with formalin (1%). Rats received a subcutaneous (s.c.) injection into the dorsal surface of the paw of vehicles or increasing doses of nalbuphine (50-200 µg/paw) or buprenorphine (1-5 µg/paw) 20 min before formalin injection into the paw. Nalbuphine antinociception was reversed by the s.c. injection into the paw of the inhibitor of NO synthesis (NG-nitro-l-arginine methyl ester (L-NAME)), by the inhibitor of guanylyl cyclase (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ)), by the Kir6.1-2, ATP-sensitive K+ channel inhibitors (glibenclamide and glipizide), by the KCa2.1-3, small conductance Ca2+-activated K+ channel blocker (apamin), by the KCa1.1, large conductance Ca2+-activated K+ channel blocker (charybdotoxin), and by the KV, voltage-dependent K+ channel inhibitors (4-aminopyridine (4-AP) and tetraethylammonium chloride (TEA)). The antinociceptive effect produced by buprenorphine was blocked by the s.c. injection of 4-AP and TEA but not by L-NAME, ODQ, glibenclamide, glipizide, apamin, or charybdotoxin. The present results provide evidence for differences in peripheral mechanisms of action between these opioid drugs.

Inhibition of ATP-sensitive potassium channels exacerbates anoxic coma in Locusta migratoria.

Under extreme environmental conditions, many insects enter a protective coma associated with a spreading depolarization (SD) of neurons and glia in the central nervous system (CNS). Recovery depends on the restoration of ion gradients by mechanisms that are not well understood. We investigated the effects of glybenclamide, an ATP-sensitive K+ (KATP) channel inhibitor, and pinacidil, a KATP activator, on the mechanisms involved in anoxic coma induction and recovery in Locusta migratoria. KATP channels allow for the efflux of K+ when activated, thereby linking cellular metabolic state to membrane potential. In intact locusts, we measured the time to enter a coma after water immersion and the time to recover the righting reflex after returning to normoxia. In semi-intact preparations, we measured the time to SD in the metathoracic ganglion after flooding the preparation with saline or exposing it to 100% N2 gas, and the time for the transperineurial potential to recover after removal of the saline or return to air. Glybenclamide decreased the time to coma induction, whereas pinacidil increased induction times. Glybenclamide also lengthened the time to recovery and decreased the rate of recovery of transperineurial potential after SD. These results were not the same as the effects of 10-2 M ouabain on N2-induced SD. We conclude that glybenclamide affects the CNS response to anoxia via inhibition of KATP channels and not an effect on the Na+/K+-ATPase.NEW & NOTEWORTHY We demonstrate the involvement of ATP-sensitive K+ (KATP) channels during recovery from spreading depolarization (SD) induced via anoxic coma in locusts. KATP inhibition using glybenclamide impaired ion homeostasis across the blood-brain barrier resulting in a longer time to recovery of transperineurial potential following SD. Comparison with ouabain indicates that the effects of glybenclamide are not mediated by the Na+/K+-ATPase but are a result of KATP channel inhibition.

The role of KATP channel blockade and activation in the protection against neurodegeneration in the rotenone model of Parkinson's disease.

AIMS: Several studies suggested that ATP-sensitive potassium channels (KATP) are potential therapeutic targets for protection against various neurodegenerative disorders, yet, there is an ongoing controversy regarding their role in Parkinson's disease (PD). Thus, the aim of the current study is to investigate the protective effect of KATP blockade and activation in the mice rotenone model of PD. MAIN METHODS: PD has been induced by 9 subcutaneous injections of rotenone (1.5 mg/kg; 3 times/week) in adult male Swiss albino mice. For 3 consecutive weeks, parkinsonian mice were either untreated or treated with L-dopa (25 mg/kg), the KATP channel blocker glibenclamide (3 mg/kg) or the KATP channel opener nicorandil (6 mg/kg). KEY FINDINGS: Glibenclamide significantly improved motor performance in the wire hanging and stair tests and halted the decline in striatal dopamine content as well as dopaminergic neurons' density. In addition, it reduced the rotenone-induced apoptosis as portrayed in the immunohistopathological examination via increasing Bcl-2 and decreasing caspases-3, -8, -9 contents. Furthermore, through its anti-inflammatory potential, glibenclamide reduced tumor necrosis factor-alpha level. On the other hand, nicorandil failed to mitigate the rotenone-induced neurodegenerative consequences. SIGNIFICANCE: KATP channel blockade by glibenclamide has neuroprotective effect against rotenone-induced neurotoxicity, that was mediated by its anti-inflammatory effect along with hindering apoptosis through extrinsic and intrinsic pathways.

Dextromethorphan and Dextrorphan Influence Insulin Secretion by Interacting with KATP and L-type Ca2+ Channels in Pancreatic ß-Cells.

The NMDA receptor antagonist dextromethorphan (DXM) and its metabolite dextrorphan (DXO) have been recommended for treatment of type 2 diabetes mellitus because of their beneficial effects on insulin secretion. This study investigates how different key points of the stimulus-secretion coupling in mouse islets and ß-cells are influenced by DXM or DXO. Both compounds elevated insulin secretion, electrical activity, and [Ca2+]c in islets at a concentration of 100 µM along with a stimulating glucose concentration. DXO and DXM increased insulin secretion approximately 30-fold at a substimulatory glucose concentration (3 mM). Patch-clamp experiments revealed that 100 µM DXM directly inhibited KATP channels by about 70%. Of note, DXM decreased the current through L-type Ca2+ channels about 25%, leading to a transient reduction in Ca2+ action potentials. This interaction might explain why elevating DXM to 500 µM drastically decreased insulin release. DXO inhibited KATP channels almost equally. In islets of KATP channel-deficient sulfonylurea receptor 1 knockout mice, the elevating effects of 100 µM DXM on [Ca2+]c and insulin release were completely lost. By contrast, 100 µM DXO still increased glucose-stimulated insulin release around 60%. In summary, DXM-induced alterations in stimulus-secretion coupling of wild-type islets result from a direct block of KATP channels and are partly counteracted by inhibition of L-type Ca2+ channels. The stimulatory effect of DXO seems to be based on a combined antagonism on KATP channels and NMDA receptors and already occurs under resting conditions. Consequently, both compounds seem not to be suitable candidates for treatment of type 2 diabetes mellitus. SIGNIFICANCE STATEMENT: This study shows that the use of dextromethorphan as an antidiabetic drug can cause unpredictable alterations in insulin secretion by direct interaction with KATP and L-type Ca2+ channels besides its actual target, the NMDA receptor.

Physiological function and molecular composition of ATP-sensitive K+ channels in human gastric smooth muscle.

Gastric motility is controlled by slow waves. In general, the activation of the ATP-sensitive K+ (KATP) channels in the smooth muscle opposes the membrane excitability and produces relaxation. Since metabolic inhibition and/or diabetes mellitus are accompanied by dysfunctions of gastric smooth muscle, we examined the possible roles of KATP channels in human gastric motility. We used human gastric corpus and antrum smooth muscle preparations and recorded the mechanical activities with a conventional contractile measuring system. We also identified the subunits of the KATP channels using Western blot. Pinacidil (10 µM), a KATP channel opener, suppressed contractions to 30% (basal tone to -0.2 g) of the control. The inhibitory effect of pinacidil on contraction was reversed to 59% of the control by glibenclamide (20 µM), a KATP channel blocker. The relaxation by pinacidil was not affected by a pretreatment with L-arginine methyl ester, tetraethylammonium, or 4-aminopyridine. Pinacidil also inhibited the acetylcholine (ACh)-induced tonic and phasic contractions in a glibenclamide-sensitive manner (42% and 6% of the control, respectively). Other KATP channel openers such as diazoxide, cromakalim and nicorandil also inhibited the spontaneous and ACh-induced contractions. Calcitonin gene-related peptide (CGRP), a gastric neuropeptide, induced muscle relaxation by the activation of KATP channels in human gastric smooth muscle. Finally, we have found with Western blot studies, that human gastric smooth muscle expressed KATP channels which were composed of Kir 6.2 and SUR2B subunits.

Glibenclamide restores dopaminergic reward circuitry in obese mice through interscauplar brown adipose tissue.

BACKGROUND: Obesity, a critical feature in metabolic disorders, is associated with medical depression. Recent evidence reveals that brown adipose tissue (BAT) activity may contribute to mood disorders, Adenosine triphosphate (ATP)-sensitive K+ (KATP) channels regulate BAT sympathetic nerve activity. However, the mechanism through which BAT activity affects mood control remains unknown. We hypothesized the BAT is involved in depressive-like symptoms regulation by trafficking KATP channels. METHODS: Eight-week-old male B6 mice fed with a high-fat diet (HFD) for 12 weeks exhibited characteristics of metabolic disorders, including hyperglycemia, hyperinsulinemia, and hyperlipidemia, as well as depressive symptoms. In this study, we surgically removed interscapular BAT in mice, and these mice exhibited immobility in the forced swim test and less preference for sugar water compared with other mice. To delineate the role of KATP channels in BAT activity regulation, we implanted a miniosmotic pump containing glibenclamide (GB), a KATP channel blocker, into the interscapular BAT of HFD-fed mice. RESULTS: GB infusion improved glucose homeostasis, insulin sensitivity, and depressive-like symptoms. KATP channel expression was lower in HFD-fed mice than in chow-fed mice. Notably, GB infusion in HFD-fed mice restored KATP channel expression. CONCLUSION: KATP channels are functionally expressed in BAT, and inhibiting BAT-KATP channels improves metabolic syndromes and reduces depressive symptoms through beta-3-adrenergic receptor-mediated protein kinase A signaling.

ATP sensitive potassium (KATP) channel inhibition: A promising new drug target for migraine.

BACKGROUND: Recently, the adenosine triphosphate (ATP) sensitive potassium channel opener levcromakalim was shown to induce migraine attacks with a far higher incidence than any previous provoking agent such as calcitonin gene-related peptide. Here, we show efficacy of ATP sensitive potassium channel inhibitors in two validated rodent models of migraine. METHODS: In female spontaneous trigeminal allodynic rats, the sensitivity of the frontal region of the head was tested by an electronic von Frey filament device. In mice, cutaneous hypersensitivity was induced by repeated glyceryl trinitrate or levcromakalim injections over nine days, as measured with von Frey filaments in the hindpaw. Release of calcitonin gene-related peptide from dura mater and trigeminal ganglion was studied ex vivo. RESULTS: The ATP sensitive potassium channel inhibitor glibenclamide attenuated the spontaneous cephalic hypersensitivity in spontaneous trigeminal allodynic rats and glyceryl trinitrate-induced hypersensitivity of the hindpaw in mice. It also inhibited CGRP release from dura mater and the trigeminal ganglion isolated from spontaneous trigeminal allodynic rats. The hypersensitivity was also diminished by the structurally different ATP sensitive potassium channel inhibitor gliquidone. Mice injected with the ATP sensitive potassium channel opener levcromakalim developed a progressive hypersensitivity that was completely blocked by glibenclamide, confirming target engagement. CONCLUSION: The results suggest that ATP sensitive potassium channel inhibitors could be novel and highly effective drugs in the treatment of migraine.

ATP-sensitive K+ channel inhibition in rats decreases kidney and skeletal muscle blood flow without increasing sympathetic nerve discharge.

ATP-sensitive K+ (KATP) channels contribute to exercise-induced hyperemia in skeletal muscle either locally by vascular hyperpolarization or by sympathoinhibition and decreased sympathetic vasoconstriction. However, mean arterial pressure (MAP) regulation via baroreceptors and subsequent efferent activity may confound assessment of vascular versus neural KATP channel function. We hypothesized that systemic KATP channel inhibition via glibenclamide (GLI) would increase MAP without increasing sympathetic nerve discharge (SND). Lumbar and renal nerve SND were measured in anesthetized male rats with intact baroreceptors (n = 12) and sinoaortic denervated (SAD; n = 4) counterparts and blood flow (BF) and vascular conductance (VC) assessed in conscious rats (n = 6). GLI increased MAP (p < 0.05) and transiently decreased HR in intact (p < 0.05), but not SAD rats. Renal (-30 %) and lumbar (-40 %) ΔSND decreased in intact but increased in SAD rats (∼40 % and 20 %; p < 0.05). BF and VC decreased in kidneys and total hindlimb skeletal muscle (p < 0.05). Thus, because KATP inhibition decreases SND, GLI-induced reductions in blood flow cannot result from enhanced sympathetic activity.

Glibenclamide inhibits BK polyomavirus infection in kidney cells through CFTR blockade.

BK polyomavirus (BKPyV) is a ubiquitous pathogen in the human population that is asymptomatic in healthy individuals, but can be life-threatening in those undergoing kidney transplant. To-date, no vaccines or anti-viral therapies are available to treat human BKPyV infections. New therapeutic strategies are urgently required. In this study, using a rational pharmacological screening regimen of known ion channel modulating compounds, we show that BKPyV requires cystic fibrosis transmembrane conductance regulator (CFTR) activity to infect primary renal proximal tubular epithelial cells. Disrupting CFTR function through treatment with the clinically available drug glibenclamide, the CFTR inhibitor CFTR172, or CFTR-silencing, all reduced BKPyV infection. Specifically, time of addition assays and the assessment of the exposure of VP2/VP3 minor capsid proteins indicated a role for CFTR during BKPyV transport to the endoplasmic reticulum, an essential step during the early stages of BKPyV infection. We thus establish CFTR as an important host-factor in the BKPyV life cycle and reveal CFTR modulators as potential anti-BKPyV therapies.